HIV-1 Intasome Assembly and Function

HIV-1 整合体的组装和功能

基本信息

批准号：
10767386
负责人：
KRISTINE E YODER
金额：
$ 31.47万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-12-15 至 2028-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10767386
关键词：
Acquired Immunodeficiency Syndrome Address Affect Architecture Binding Biology Biophysics Chimera organism Chromatin Chromosomes Clinical Collaborations Complex Costs and Benefits DNA DNA Repair Disease Drug Targeting Drug resistance Exhibits Family member Genome Genomics Goals HIV HIV-1 HIV-1 integrase Histones Human Human Genome In Vitro Individual Infection Integrase Integration Host Factors Kinetics Length Lentivirus Lesion Link Long Terminal Repeats Mechanics Mouse Mammary Tumor Virus Mutation Nuclear Envelope Nucleosomes Outcome Pathogenicity Pattern Peptides Positioning Attribute Post-Translational Protein Processing Process Productivity Proteins Regimen Reporting Retroviridae Reverse Transcription Role Side Site Spumavirus Structure Time Viral Virus animation antiretroviral therapy cellular transduction cofactor combinatorial design dimer feature detection flexibility genomic RNA imaging platform in vitro activity innovation integration site leukemia molecular imaging pandemic disease particle prototype resistance mutation single molecule stoichiometry therapeutic target transcriptional coactivator p75 vector

项目摘要

PROJECT SUMMARY / ABSTRACT New HIV-1 infections continue to drive a worldwide pandemic. Combinatorial anti-retroviral therapies (cART) have helped to blunt the clinical outcomes of HIV-1 infected individuals. However, drug-resistance mutations continue to challenge cART regimens underscoring the importance of identifying new viral drug targets. HIV-1 integration into the human genome is essential for a productive infection. Integration is catalyzed by the retrovirus encoded integrase (IN) that forms a complex with the long terminal repeat (LTR) ends of the viral cDNA, produced by reverse transcription of the HIV-1 genomic RNA. The resulting intasome precisely positions the LTR-ends for catalytic strand-transfer into a genomic target site. Structural comparisons show that all seven retrovirus genera maintain a central Conserved Intasome Core (CIC) containing a tetramer of IN. Some retrovirus family members expand the structure surrounding the CIC by appending additional IN subunits. For example, the prototype foamy virus (PFV) intasome assembles into a simple IN-tetramer while the mouse mammary tumor virus (MMTV) forms an IN-octamer by attaching IN-dimers to either side of the CIC. IN octamer, decamer, dodecamer (12-mer) and hexadecamer (16-mer) intasomes have been reported for HIV-1. Remarkably, the contributions of IN-multimer architecture to HIV-1 biology is largely unknown. The IN-assembly progressions that result in a fully formed HIV-1 intasome are similarly unknown. During infection, reverse transcription and intasome assembly occurs at or near the nuclear membrane. The host cofactor LEDGF/p75 appears to facilitate chromatin localization of the HIV-1 intasomes, and deletion of LEDGF/p75 reduces HIV-1 integration at least 10-fold. We have found the non-conserved peptides linking well- known conserved IN domains control HIV-1 IN-multimer architecture, and that LEDGF/p75 is necessary for efficient HIV-1 intasome assembly in vitro. These observations underpin several key unanswered questions: What are the factors that guide IN multimer progressions resulting in a fully assembled HIV-1 intasome? What is the function of LEDGF/p75 in HIV-1 intasome assembly and/or chromatin interactions? How does HIV-1 IN- multimer architecture impact genomic target site selection in cellulo? We propose to utilize innovative real-time single molecule imaging and analysis to understand the contributions of IN-multimer architecture on HIV-1 mechanics with the following Specific Aims: 1.) determine the IN assembly progressions that control HIV-1 intasome architecture, 2.) determine the role of HIV-1 intasome architecture on the dynamic interactions with defined target DNA and chromatin in vitro, and 3.) determine the role of HIV-1 intasome architecture on targeting host chromatin features in cellulo. These studies are designed to interrogate the animated processes that support HIV-1 intasome architecture with the goal of identifying additional retroviral progressions that might be exploited as therapeutic targets.

项目概要/摘要新的 HIV-1 感染继续导致全球大流行。联合抗逆转录病毒疗法（cART）有助于削弱 HIV-1 感染者的临床结果。然而，耐药突变继续挑战 cART 方案，强调识别新病毒药物靶点的重要性。 HIV-1 整合到人类基因组中对于有效感染至关重要。整合的催化因素是逆转录病毒编码的整合酶（IN）与病毒的长末端重复（LTR）末端形成复合物 cDNA，由 HIV-1 基因组 RNA 逆转录产生。所得的嵌体精确定位用于催化链转移到基因组靶位点的 LTR 末端。结构比较表明，所有七个逆转录病毒属维持包含 IN 四聚体的中央保守整合体核心 (CIC)。一些逆转录病毒家族成员通过附加额外的 IN 亚基来扩展 CIC 周围的结构。为了例如，原型泡沫病毒（PFV）整合体组装成简单的IN四聚体，而小鼠乳腺肿瘤病毒 (MMTV) 通过将 IN-二聚体连接到 CIC 的任一侧来形成 IN-八聚体。在八聚体中，据报道，HIV-1 存在十聚体、十二聚体（12 聚体）和十六聚体（16 聚体）嵌体。值得注意的是，IN 多聚体结构对 HIV-1 生物学的贡献在很大程度上尚不清楚。导致完全形成的 HIV-1 嵌体的 IN 组装进程同样未知。期间感染、逆转录和嵌体组装发生在核膜处或核膜附近。主人辅助因子 LEDGF/p75 似乎促进 HIV-1 嵌体的染色质定位，并删除 LEDGF/p75 使 HIV-1 整合减少至少 10 倍。我们发现非保守肽连接良好- 已知的保守 IN 结构域控制 HIV-1 IN 多聚体结构，并且 LEDGF/p75 对于 HIV-1 IN 多聚体结构是必需的体外有效的 HIV-1 嵌体组装。这些观察结果支撑了几个尚未解答的关键问题：引导 IN 多聚体进展产生完全组装的 HIV-1 嵌体的因素有哪些？什么 LEDGF/p75 在 HIV-1 嵌体组装和/或染色质相互作用中的功能是什么？ HIV-1如何进入- 多聚体结构影响细胞中基因组靶位点的选择？我们建议利用创新的实时单分子成像和分析来了解其贡献 HIV-1 机制上的 IN 多聚体架构的研究，具有以下具体目标：1.) 确定 IN 组装控制 HIV-1 嵌体结构的进展，2.) 确定 HIV-1 嵌体结构在体外与确定的目标 DNA 和染色质的动态相互作用，以及 3.) 确定 HIV-1 的作用细胞内靶向宿主染色质特征的整合体结构。这些研究旨在探究支持 HIV-1 嵌体结构的动画过程目的是确定可能被用作治疗靶点的其他逆转录病毒进展。